mmg_233_2013_genetics_genomicswikiaorg-20200214-history
C. elegans: microRNA
Caenorhabditis elegans have been used since the 1980's to discover small RNA structures within the organism. In 1993, Lee et al., discovered microRNA's in C. elegans through cloning by complementation and the comparison of the RNA:RNA interactions of lin-4 and lin-14 RNAs [1]. MicroRNA's are non-protein coding RNAs the regulate gene expression through transcriptional and post-transcriptional interactions with mRNA [1]. Lee et al. found that the lin-4 microRNA interactes with lin-14 mRNA thereby decreasing the LIN-14 protein during the C. elegans embryonic development 3. The Discovery of microRNA's lin-4 Locus: Lee et al., discovered, after cloning by complementation, that there was a lin-4 gene in the C. elegans ''organism. He did this by isolating a ''C. elegans that presented the mutation of interest. Plasmids that had different fragments of the C. elegans DNA were added to the mutated C. elegans. One of these plasmid fragments will contain the wild type DNA that will correct the gene mutation in the C. elegans. After incubation the wild type that corrects the mutation can be isolated and the DNA sequence of that wild-type can be compared to the DNA of the C. elegans with the mutation 2. Lee et al. found that there was a 693 bp sequences that rescued this loss of function lin-4 mutation. His group then compared this sequence to a bunch of allele sequences that were known to code for proteins, and found that it was epistatic to the sequence of the lin-14 alleles 3. lin-4 is Unlikely to Encode a Protein: There are two reasons that Lee et al. believe that the lin-4 allele does not code for a protein. First, when they had placed frameshift mutations, nonsense mutation, and ATG to ACG mutations into open reading frames it did not affect the rescuing activity of the lin-4 loss of function mutation 3. Second, when they compared C. elegans to other Caenorhabditis species, which were functionally interchangeable with the C. elegans, they found that DNA sequences were conserved however open reading frames were not. If the lin-4 were to code for a protein then its synthesis would have to be directed by start and stop codons 3. lin-4 Encodes Small RNAs: lin-4S RNA and lin-4L RNA are considered to be gene products of lin-4. Lee et al. ran a Northern blot and were able to detect these small RNA fragment in both the wild-type plasmid and the rescued lin-4 mutated plasmid. They also found that the lin-4S and lin-4L RNA fragment had the same sequence in all of the Caenorhabditis species. Besides finding fragment of the small RNAs only when a functional lin-4 was present; they also found that the promotor region of the lin-4S and lin-4L at the 5' region were identical and matched up with the 3' UTR of the lin-14 mRNA3. Antisense Complementarity to the lin-14 3'UTR It was found the lin-4 is an antisense RNA that down regulates lin-14. They believed this for three reasons. First, when they compared different species of Caenorhabditis the lin-4:lin-14 base pairing was conserved between the species. Second, the loss of function mutation for the lin-4 was within the lin-14 sequence. Finally, seven lin-4 elements are complementary to a lin-14 region that are deleted by a lin-14 gain of function allele. These three reasons are why Lee et al. believe that lin-4 down regulates LIN-14 protein levels through RNA:RNA interaction at the lin-14 3'UTR sight 3. Temporal Regulation of lin-14 by lin-4: If it true the lin-14 is inhibited by lin-4 at the 3' UTR of lin-14 there would be a temporal decrease of the LIN-14 protein. In the early stages of C. elegans development lin-4 is inactive and there is a high level of LIN-14 protein. It is believed that food may trigger the signal for lin-4 to activate lin-4S and lin-4L gene transcripts because LIN-14 protein only decreases after feeding, which is between the L1 and L2 stage of development 3. Antisense Regulaton via 3'UTR's: 'l'in-4 does not seem to effect the stability or the translatability of lin-14 mRNA because throughout the entire development of the C. elegans lin-14 mRNA levels remain constant. Lee et al. is not sure how exactly lin-4 inhibits lin-14 but it seems like it does not degrade it like other forms of microRNA's can. They assume lin-4 inhibits the production of LIN-14 protein by modifying the lin-14 mRNA, or by localizing the lin-14 mRNA into compartments in the nucleus so ribosomes can not access the mRNA, or lin-4 could bind to the mRNA in the cytoplasm interacting directly with translational machinery. They could interact with this other machinery by the involvement of the loops that are formed by areas of lin-4 microRNA that do not compliment the lin-14 mRNA in the lin-4:lin-14 interaction 3. Small Regulatory RNAs: When this study was released lin-4S and lin-4L microRNAs were the smallest regulatory RNA that had been discovered. lin-4S and lin-4L are only 61 and 22 bases respectively, and due to their small size they could only interact directly with very few proteins 3. References 1. MicroRNA, Wikipedia, 2013 Link: http://en.wikipedia.org/wiki/MicroRNA 2. Watson J.D., Gilman M., Witkowski J., Zoller M., (1992) Recombinant DNA. 2nd ed. New York: Scientific American Books. 3. Lee R.C., Feinbaum R. L., Ambros V., (1993) The C. elegans Heterochronic Gene lin-4 Encodes Small RNAs with Antisense Complementarity to lin-14. Cell. 75: 843-854. Link: http://www.sciencedirect.com/science/article/pii/009286749390529Y